In the present study an experimental high-altitude intestinal barrier injury rat model was established by simulating an acute hypoxia environment, to provide an experimental basis to assess the pathogenesis, prevention and treatment of altitude sickness. A total of 70 healthy male Sprague-Dawley rats were divided into two groups: Control group (group C) and a high-altitude hypoxia group (group H). Following 2 days adaptation, the rats in group H were exposed to a simulated 4,000-m, high-altitude hypoxia environment for 3 days to establish the experimental model. To evaluate the model, bacterial translocation, serum lipopolysaccharide level, pathomorphology, ultrastructure and protein expression in rats were assessed. The results indicate that, compared with group C, the rate of bacterial translocation and the apoptotic index of intestinal epithelial cells were significantly higher in group H (P<0.01). Using a light microscope it was determined that the intestinal mucosa was thinner in group H, there were fewer epithelial cells present and the morphology was irregular. Observations with an electron microscope indicated that the intestinal epithelial cells in group H were injured, the spaces among intestinal villi were wider, the tight junctions among cells were open and lanthanum nitrate granules (from the fixing solution) had diffused into the intestinal mesenchyme. The expression of the tight junction protein occludin was also decreased in group H. Therefore, the methods applied in the present study enabled the establishment of a stable, high-altitude intestinal barrier injury model in rats.